Table of Contents
What is Black Cumin Seed Oil?
Nigella sativa (Family Ranunculacae), more commonly known as back cumin seed or kalonji, is a medicinal plant that has been used around the world since antiquity. It is used in traditional medical systems such as Ayurveda, Unanai and Siddha (1). The Persian polymath, Avicenna, believed that the seeds help people recover from fatigue and called the seeds ‘The Canon Of Medicine’.
The seeds are known as ‘Habbatul barakah’ in Arabic which means ‘seeds of blessing’. The seeds are also mentioned in a number of religious texts where they are recommended as a cure for everything except death (2).
The plant is native to the Middle East and south west Asia. The plant grows to a height of 20 to 90 cm tall with colored petals and small dicotyledonous seeds. The seeds are the source of the medicinal properties of the plant. The key active ingredient in the seeds in thymoquinone (30% – 48%) (11).
The seeds can be eaten whole or pressed to make an oil extract. The oil can be taken orally or topically and has even been given intravenously in animal experiments.
Black cumin seed oil is used for a wide range of ailments and as such has been called the ‘miracle herb’. The question is whether modern science supports the accolade of ‘miracle herb’
Does Black Cumin Seed Oil Help Asthma?
There are three key clinical studies evaluating the use of black cumin seed oil for asthma.
Two of the studies were randomized placebo controlled trials in adults with asthma (10). Both studies showed statistically significant improvements in asthma symptoms in patients receiving Nigella. One of the studies showed significant improvements in lung function tests and a decrease in the usage of inhalers and oral medication as compared to the control group (10).
The results of the second study were different and showed only a trend towards an improvement in lung function tests in the Nigella treatment group (Javan & Koshak). The second study did show that patients treated with Nigella had normalization of their peripheral blood eosinophils count. Eosinophils are a subset of the white cell count and raised eosinophil counts are associated with allergic conditions such as asthma.
The third study population was different but relevant to inflammatory lung disorders.
Forty chemical war victims were randomly allocated to receive either boiled extract of Nigella seed or placebo for two months (10). Significant improvements in lung function were noted in the treatment arm as compared to the control arm during the study.
From a medical perspective, studies like these that involve less than 100 patients are considered to be ‘pilot’ or ‘proof of concept studies’. They are not definitive or conclusive studies from which treatment guidelines can be made. This is especially the case when the results of pilot studies are conflicting. Positive ‘pilot’ or ‘proof of concept studies’ usually lead to larger clinical trials which are needed in order to inform clinical decision making.
In summary, there are positive signals from early clinical studies to support a possible role for black cumin seed oil as an adjunctive treatment in asthma. This means that black cumin seed could be considered in future clinical trials as an add-on to standard therapy. There is no evidence to support the use of black cumin seed oil instead of standard medical therapy.
Does It help Eczema?
There is a single clinical study looking at the effects of black cumin seed oil for eczema (27). Sixty patients with hand eczema were randomly allocated to either topical steroids, black cumin seed or a commercially available eczema brand. Study subjects applied the allocated cream twice daily for one month.
The patients receiving black cumin seed or steroids showed a greater improvement in their hand eczema as compared to the group receiving the commercial skin care brand. No significant difference was noted between the black cumin seed arm and the steroid arm of the study.
In summary, this study suggests that black cumin seed oil might be as effective as topical steroids for hand eczema but again the study is limited by the small number of patients and the short duration of follow-up.
Does It help digestion and Helicobacter pylori eradication?
There are two key clinical studies which have looked at black cumin seed oil for patients with dyspepsia. These two clinical studies had very different designs. Each study tells a different story but gives us unique insights into black cumin seed oil which adds to our overall understanding of this herb.
The first study comes from Tehran, was published in 2015 and evaluated the effect of a honey based formulation of black cumin seed oil on dyspepsia (23).
Seventy patients with dyspepsia were randomly allocated to receive a traditional honey based formulation of black cumin seed oil (5 ml) or placebo in addition to a proton pump inhibitor (antacid) for eight weeks.
The study showed that eight weeks of supplementation with a honey based formulation of black cumin seed oil improved symptoms of dyspepsia, the rate of Helicobacter pylori eradication and overall quality of life. No serious adverse events were reported during the study.
The authors acknowledged that the short duration of the study limited the interpretation of the results of this study given the fact that dyspepsia is a chronic condition. The authors recommended that studies with longer term follow-up should be done.
A second study was done in Saudi Arabia and looked at the effect of black cumin seed for the eradication of Helicobacter pylori in patients with non-ulcer dyspepsia. Helicobacter pylori is associated with dyspepsia and is usually treated with triple therapy which consists of a combination of two antibiotics (amoxicillin and clarithromycin) plus a proton pump inhibitor (omeprazole).
In this study, 88 patients with Helicobacter positive dyspepsia were allocated to four groups: standard triple therapy, Nigella sativa 1gm plus omeprazole, Nigella sativa 2 gm plus omeprazole or Nigella sativa 3 gm plus omeprazole.
Dyspepsia symptoms improved in all study groups to a similar extent. The study found that Helicobacter pylori eradication was not statistically different between the triple therapy arm and the 2 gm plus omeprazole arm. However Nigella sativa 1gm plus omeprazole or Nigella sativa 3 gm plus omeprazole were significantly less effective than standard triple therapy. It is unclear why different doses of Nigella sativa would produce different results.
Taken together these two studies show us the risks of just taking the results of a single positive pilot study to inform clinical decision making. The first study suggested that black cumin seed oil was helpful for dyspepsia but the second study showed that the effect of black cumin seed varies depending on the dose used.
In summary, larger longer term studies are needed before any firm conclusions can be made about the role and optimal dose of black cumin seed for dyspepsia.
Does It help Candida?
There are no human clinical studies looking at the role of black cumin seed oil in candida infections. A laboratory based study in India showed that black cumin seed oil extract showed anifungal activity in vitro against the four species of candida that are pathogenic in humans (24).
However this has not yet been extended into the clinical research arena in humans.
In summary, there is no human data to support the role of black cumin seed for fungal infections such as candida.
Does It Affect Cancers?
Hundreds of papers have been published on the anti-cancer potential of thymoquinone. A review of the current status of research in this area was published last month (19). The review discussed the potential of thymoquinone as an anticancer molecule, its mechanism of action and future usage in clinical applications.
The paper explains that thymoquinone exhibits anticancer activity via numerous mechanisms of action, specifically by showing selective antioxidant and oxidant activity, interfering with DNA structure, affecting carcinogenic signalling molecules and immunomodulation.
The authors add that in vitro activity of thymoquinone has been further implicated in animal models of cancer; however, no clinical application has been proven yet’.
The overall conclusion of the review is that ‘This is the optimum time to focus on clinical trials for developing thymoquinone as a future drug in cancer therapeutics’.
In summary, there are no clinical studies to recommend the use of black cumin seed for cancer.
Does It Promote heart health?
There are no studies looking at real life heart health clinical outcomes with black cumin seed oil. Instead there are a number of studies looking at surrogate or indirect markers of heart health such as blood pressure and lipids.
Two studies from Pakistan produced conflicting data on the effect of Nigella on lipid profiles. In the first study, Nigella sativa seed supplementation resulted in no statistically significant difference on serum lipids, blood sugar, blood pressure and body weight in a randomized double blind study of 123 patients in Karachi (21).
This contrasts with the results of another study done in Karachi which compared black cumin seeds (1.5 gm /day) alone versus turmeric (2.4 gm/ day) alone versus the combination of black cumin seeds (0.9 gm /day) plus turmeric (1.5 gm/ day) versus placebo (4). Two hundred and fifty males who screened positive for the metabolic syndrome were enrolled in the study over an eight week period.
At the end of the study, compared to placebo, improvements in body fat, fasting blood glucose, cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol were noted in the turmeric plus black seed combination arm. This is interesting considering the fact that the combination arm had only 60% of the dose of the individual herbs.
Hence, it is impossible to rule out the fact that turmeric was the key active ingredient in this study or that black cumin has a sweet spot in terms of optimal dosing.
A third study looked at the effect of 2 gm of black cumin seed daily for 4 weeks in 88 patients with dyslipidemia in Iran (25). Significant decreases in low density lipoproteins, triglycerides and total cholesterol were noted in the treatment as compared to the placebo arm of the study. No benefits were noted on fasting blood sugar or high density lipoproteins.
It is simply not possible to synthesize the data and draw overarching conclusions from the results of the studies done in Pakistan and Iran because the results are so different across the studies.
Another key risk factor for heart disease is hypertension. The effects of Nigella sativa at doses of 200mg or 400mg daily or placebo on patients with mild hypertension was studied over a period of eight weeks in Shahrekord, Iran (7). The study showed that daily use of black cumin seed resulted in a statistically significant reduction in blood pressure.
Of note, this finding on BP was not replicated in the first study from Pakistan mentioned above (21).
In summary, the results of a number of studies evaluating black cumin seed extract on indirect measures of heart health are inconclusive and conflicting. Small sample sizes, short duration of follow-up, different doses of Nigella sativa and different study designs likely contributed to the confusion.
Does it promote immune health?
The potential role of thymoquinone as an anti-oxidant, anti-inflammatory and immunomodulator has been the subject of numerous publications. However human clinical data are limited to four studies relevant to immune health.
Significant decreases in disease activity score was noted in 40 female patients with rheumatoid arthritis who took placebo for one month followed by Nigella sativa 500mg twice daily for one month (11). The patients continued on their usual medications during the study. The authors conduced that Nigella sativa may be a useful adjunct therapy in patients with rheumatoid arthritis.
In the second study, Nigella sativa supplementation at a dose of 2gm per day was noted to improve CD8 cell counts in patients with allergic rhinitis (17). The patients were randomized to receive either standard immunotherapy plus placebo or standard immunotherapy plus Nigella sativa.
In the third paper, four studies on the clinical efficacy of Nigella sativa in allergic diseases were presented. In these studies, a total of 152 patients with allergic diseases (allergic rhinitis, bronchial asthma, atopic eczema) were treated with Nigella sativa oil, given in capsules at a dose of 40 to 80 mg/kg/day (18). Patients reported improvements in the subjective symptoms of allergy but this was not supported by any changes in laboratory parameters. This study had no placebo arm.
The fourth paper looked at topical application of black seed oil in the treatment of allergic rhinitis (3). A total of 68 patients with allergic rhinitis were randomized to either Nigella sativa nasal drops or placebo oil. At the end of 6 weeks, the improvement in tolerability of allergen exposure in active group was 55.2% as compared with control group of 20%.
A comprehensive review of the immunomodulatory and anti-inflammatory action of Nigella sativa and thymoquinone in 2015 concluded that ‘experimental evidence suggests that N. sativa extracts can potentially be employed in the development of effective therapeutic agents towards the regulation of immune reactions implicated in various infectious and non-infectious conditions including different types of allergy, autoimmunity, and cancer’. (Majdalawieh). There has been no new studies in the interim to change the conclusion of this overview.
In summary, experimental data suggests that Nigella may play a role in a range of immune based disorders but there is essentially not enough information available to recommend its use at this time.
Does it help Skin and hair?
There is very little information available on the use of Nigella sativa for hair or nails. A single study in humans showed that Nigella sativa oil improved vitiligo in 52 patients who were randomized to receive either Nigella sativa or placebo in addition to their usual treatment for vitiligo (13).
Laboratory based studies suggest a possible role for black cumin seed oil in psoriasis but this has not yet been studied in humans (8).
In summary, black cumin seed oil cannot be recommended for hair or skin at this time.
Does Black Cumin Seed Oil help MRSA?
There is growing interest in the use of essential oils as antimicrobial agents. It is believed that the essential oils can disrupt the lipid bilayer of bacteria.There is particular interest in the possible role of Nigella sativa in the treatment of drug resistant bacteria (5).
Thymoquinone exhibited MRSA bacterocidal properties and a post-antibiotic effect (meaning that the thymoquinone continued to kill the bacteria even after discontinuation of the thymoquinone ) for 99 strains of MRSA in a laboratory based study (Haniharan).
Aditionally, a laboratory study from Nigeria evaluated the susceptibility of multi-drug resistant Staph aureus to Nigella sativa (9). Resistant staphylococcus aureus was isolated from patients with diabetic foot ulcers. 42% of the resistant Staph aureus isolates were susceptible to Nigella sativa in the laboratory.
While the majority of MRSA strains are sensitive to vancomycin, vancomycin resistant strains of MRSA are now emerging especially in Asian countries. A total of 51 vancomycin resistant MRSA strains were tested against black cumin seed oil extract and found to be susceptible to this essential oil in vitro (20).
In summary, while Nigella sativa is a promising agent for the management of drug resistant bacteria such as MRSA, it is worth noting that there are no clinical outcome studies to support its use at this time.
Are there side effects?
Nigella sativa is well tolerated in the available published clinical studies. However as mentioned before the majority of these studies are limited by short-term follow up of small numbers of patients.
A single case report from France described a case of a patient who developed a clinically significant bullous skin rash following topical and oral use of Nigellla sativa (10).
There is very limited clinical information available on the benefits of black cumin seeds in humans. At best, available studies suggest that black cumin seed oil may be a useful adjunctive therapy for some diseases. However, well-designed studies of larger numbers of patients are needed to clarify the ideal role, optimal dose and long term safety of black cumin seed oil in humans.
Even though the existing studies fail to answer the question as to whether black cumin seed oil can promote health, each pilot study tells part of the story of Nigella sativa and could be used to design better, more informative studies in the future.
Realistically, given the fact that there are effective treatments for conditions such as dysiplidemia and asthma, really large robustly designed studies would be needed to prove that black cumin seed is ‘non-inferior’ to existing treatments.
As things stand, modern science cannot currently support the accolade of ‘miracle herb’ for Nigella sativa.
- Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, Damanhouri ZA, Anwar F. A review on therapeutic potential of Nigella sativa: A miracle herb. Asian Pac J Trop Biomed. 2013 May;3(5):337-52. doi: 10.1016/S2221-1691(13)60075-1. Review.
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- Alsamarai AM, Abdulsatar M, Ahmed Alobaidi AH. Evaluation of topical black seed oil in the treatment of allergic rhinitis. Antiinflamm Antiallergy Agents Med Chem. 2014 Mar;13(1):75-82
- Amin F, Islam N, Anila N, Gilani AH. Clinical efficacy of the co-administration of Turmeric and Black seeds (Kalongi) in metabolic syndrome – a double blind randomized controlled trial – TAK-MetS trial. Complement Ther Med. 2015 Apr;23(2):165-74. doi: 10.1016/j.ctim.2015.01.008. Epub 2015 Jan 14.
- Bakal SN, Bereswill S, Heimesaat MM.Finding Novel Antibiotic Substances from Medicinal Plants – Antimicrobial Properties of <i>Nigella Sativa</i> Directed against Multidrug-resistant Bacteria. Eur J Microbiol Immunol (Bp). 2017 Mar 16;7(1):92-98. doi: 10.1556/1886.2017.00001. eCollection 2017 Mar.
- Darakhshan S, Bidmeshki Pour A, Hosseinzadeh Colagar A, Sisakhtnezhad S.Thymoquinone and its therapeutic potentials. Pharmacol Res. 2015 May-Jun;95-96:138-58. doi: 10.1016/j.phrs.2015.03.011. Epub 2015 Mar 28. Review.
- Dehkordi FR, Kamkhah AF. Antihypertensive effect of Nigella sativa seed extract in patients with mild hypertension. Fundam Clin Pharmacol. 2008 Aug;22(4):447-52. doi: 10.1111/j.1472-8206.2008.00607.x.
- Dwarampudi LP, Palaniswamy D, Nithyanantham M, Raghu PS. Antipsoriatic activity and cytotoxicity of ethanolic extract of Nigella sativa seeds. Pharmacogn Mag. 2012 Oct;8(32):268-72. doi: 10.4103/0973-1296.103650.
- Emeka LB, Emeka PM, Khan TM. Antimicrobial activity of Nigella sativa L. seed oil against multi-drug resistant Staphylococcus aureus isolated from diabetic wounds. Pak J Pharm Sci. 2015 Nov;28(6):1985-90.
- Gelot P, Bara-Passot C, Gimenez-Arnau E, Beneton N, Maillard H, Celerier P. [Bullous drug eruption with Nigella sativa oil]. Ann Dermatol Venereol. 2012 Apr;139(4):287-91. doi: 10.1016/j.annder.2012.01.025. Epub 2012 Mar 23. French.
- Gheita TA, Kenawy SA. Effectiveness of Nigella sativa oil in the management of rheumatoid arthritis patients: a placebo controlled study. Phytother Res. 2012 Aug;26(8):1246-8. doi: 10.1002/ptr.3679. Epub 2011 Dec 12
- Gholamnezhad Z, Havakhah S, Boskabady MH. Preclinical and clinical effects of Nigella sativa and its constituent, thymoquinone: A review. J Ethnopharmacol. 2016 Aug 22;190:372-86. doi: 10.1016/j.jep.2016.06.061. Epub 2016 Jun 27. Review.
- Ghorbanibirgani A, Khalili A, Rokhafrooz D. Comparing Nigella sativa Oil and Fish Oil in Treatment of Vitiligo. Iran Red Crescent Med J. 2014 Jun;16(6):e4515. doi: 10.5812/ircmj.4515. Epub 2014 Jun 5.
- Hannan A, Saleem S, Chaudhary S, Barkaat M, Arshad MU. Anti bacterial activity of Nigella sativa against clinical isolates of methicillin resistant Staphylococcus aureus. J Ayub Med Coll Abbottabad. 2008 Jul-Sep;20(3):72-4.
- Hariharan P, Paul-Satyaseela M, Gnanamani A. In vitro profiling of antimethicillin-resistant Staphylococcus aureus activity of thymoquinone against selected type and clinical strains. Lett Appl Microbiol. 2016 Mar;62(3):283-9. doi: 10.1111/lam.12544.
- Heydari M Medicinal aspects of opium as describe in Avicenna’s Canon of Medicine. Acta Med Hist Adriat 11, 101-112.
- Işik H, Cevikbaş A, Gürer US, Kiran B, Uresin Y, Rayaman P, Rayaman E, Gürbüz B, Büyüköztürk S. Potential adjuvant effects of Nigella sativa seeds to improve specific immunotherapy in allergic rhinitis patients. Med Princ Pract. 2010;19(3):206-11. doi: 10.1159/000285289. Epub 2010 Mar 29.
- Kalus U, Pruss A, Bystron J, Jurecka M, Smekalova A, Lichius JJ, Kiesewetter H. Effect of Nigella sativa (black seed) on subjective feeling in patients with allergic diseases. Phytother Res. 2003 Dec;17(10):1209-14.
- Khan MA, Tania M, Fu S, Fu J. Thymoquinone, as an anticancer molecule: from basic research to clinical investigation. Oncotarget. 2017 Apr 18. doi: 10.18632/oncotarget.17206. [Epub ahead of print] Review.
- Liaqat F, Sheikh AA, Nazir J, Hussain T, Rabbani M, Shaheen AY, Muhammad J. Report-Isolation identification and control of vancomycin resistant Staphylococcus aureus. Pak J Pharm Sci. 2015 May;28(3):997-1004.
- Qidwai W, Hamza HB, Qureshi R, Gilani A. Effectiveness, safety, and tolerability of powdered Nigella sativa (kalonji) seed in capsules on serum lipid levels, blood sugar, blood pressure, and body weight in adults: results of a randomized, double-blind controlled trial. J Altern Complement Med. 2009 Jun;15(6):639-44. doi: 10.1089/acm.2008.0367.
- AF, Fayyad MW. Immunomodulatory and anti-inflammatory action of Nigella sativa and thymoquinone: A comprehensive review. Int Immunopharmacol. 2015 Sep;28(1):295-304. doi: 10.1016/j.intimp.2015.06.023. Epub 2015 Jun 26. Review.
- Mohtashami R, Huseini HF, Heydari M, Amini M, Sadeqhi Z, Ghaznavi H, Mehrzadi S. Efficacy and safety of honey based formulation of Nigella sativa seed oil in functional dyspepsia: A double blind randomized controlled clinical trial. J Ethnopharmacol. 2015 Dec 4;175:147-52. doi: 10.1016/j.jep.2015.09.022. Epub 2015 Sep 18.
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- Sabzghabaee AM, Dianatkhah M, Sarrafzadegan N, Asgary S, Ghannadi A. Clinical evaluation of Nigella sativa seeds for the treatment of hyperlipidemia: a randomized, placebo controlled clinical trial. Med Arch. 2012;66(3):198-200.
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- Yousefi M, Barikbin B, Kamalinejad M, Abolhasani E, Ebadi A, Younespour S, Manouchehrian M, Hejazi S. Comparison of therapeutic effect of topical Nigella with Betamethasone and Eucerin in hand eczema. J Eur Acad Dermatol Venereol. 2013 Dec;27(12):1498-504. doi: 10.1111/jdv.12033. Epub 2012 Dec 1.
Riad Agbaria, Adi Gabarin, Arik Dahan, Shimon Ben-Shabat
Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
Abstract: The traditional preparation process of Nigella sativa (NS) oil starts with roasting of the seeds, an allegedly unnecessary step that was never skipped. The aims of this study were to investigate the role and boundaries of thermal processing of NS seeds in the preparation of therapeutic extracts and to elucidate the underlying mechanism. NS extracts obtained by various seed thermal processing methods were investigated in vitro for their antiproliferative activity in mouse colon carcinoma (MC38) cells and for their thymoquinone content. The effect of the different methods of thermal processing on the ability of the obtained NS oil to inhibit the nuclear factor kappa B (NF-κB) pathway was then investigated in Hodgkin’s lymphoma (L428) cells. The different thermal processing protocols yielded three distinct patterns: heating the NS seeds to 50°C, 100°C, or 150°C produced oil with a strong ability to inhibit tumor cell growth; no heating or heating to 25°C had a mild antiproliferative effect; and heating to 200°C or 250°C had no effect. Similar patterns were obtained for the thymoquinone content of the corresponding oils, which showed an excellent correlation with the antiproliferative data. It is proposed that there is an oxidative transition mechanism between quinones after controlled thermal processing of the seeds. While NS oil from heated seeds delayed the expression of NF-κB transcription, non-heated seeds resulted in only 50% inhibition. The data indicate that controlled thermal processing of NS seeds (at 50°C–150°C) produces significantly higher anticancer activity associated with a higher thymoquinone oil content, and inhibits the NF-κB signaling pathway.
Keywords:Nigella sativa, thermal processing, antiproliferative effect, thymoquinone, NF-κB
Nigella sativa (NS), or black seed, is an annual flowering plant of the Ranunculaceae family, which is native to the Mediterranean and the neighboring countries of Pakistan and India. Although the plant is not a significant component of the human diet, in the Middle East it is incorporated into the way of life and the daily diet1 as a spice and preservative.2 Black seed has been widely used for thousands of years to treat a variety of diseases and medical conditions, including asthma, high blood pressure, diabetes, inflammation, cough, headache, eczema, fever, dizziness, and influenza.2–4 Over the last five decades, numerous scientific studies have affirmed the pharmacological qualities of NS seeds, and demonstrated its anti-inflammatory, antibacterial, antihistamine, antidiabetic, anticancer, and antihypertensive activity.2
Extracts of NS contain volatile and non-volatile oils, amino acids, proteins, carbohydrates, alkaloids, nitrogen compounds, saponins, and minerals such as sodium, calcium, iron, and potassium; overall, more than 100 compounds have been isolated from black seed and their structure elucidated.1,2 Many studies have attributed the bulk of the pharmacological activity of NS to its quinone content, which includes thymoquinone (TQ) and its dimer dithymoquinone, thymohydroquinone (THQ), and thymol, which are known for their anticancer activity. The main phytochemical component of the volatile oil of NS is TQ, which accounts for 28%–45% of the oil.2,5,6 TQ has been thoroughly studied in vitro and in vivo, and shown to have a range of therapeutic properties, including analgesic,7 antihypertensive, lipid-lowering,7–9 anti-inflammatory,10 antibacterial,11 antifungal,12 antihistaminic,13 antidiabetic,14,15 and anticancer activity.16–22 Inhibition of transcription of nuclear factor kappa B (NF-κB) has been suggested as a potential mechanism for the anticancer effects of TQ.6,23
Interviews with local traditional Bedouin herbal therapists reveal that the processing of NS seed to obtain a therapeutic oil follows a specific scheme that involves overnight soaking followed by drying, roasting for 5–6 minutes, milling, and filtration. In order to obtain only the oil from the seeds, the roasting step is not required, and the oil can be similarly obtained without this step; the fact that this allegedly unnecessary step has that has never been skipped, implies that this step may have a currently unknown role in the unique therapeutic qualities of NS oil.
The aims of this research were to investigate the role and boundaries of thermal processing of NS seed in preparation of therapeutic extracts, and to elucidate the mechanism behind the data obtained. The antiproliferative activity of NS extracts following various specifications of thermal processing of the seed were investigated in vitro in non-cancerous fibroblasts and in a mouse colon carcinoma (MC38) cell line. The effect of these thermal processing specifications on the TQ composition of the obtained NS oil was then studied and analyzed. Finally, the effect of thermal processing of the seed on the ability of the obtained NS oil to inhibit the NF-κB pathway was investigated in Hodgkin’s lymphoma (L428) cells. Overall, this protocol allowed us to identify the relationship between seed processing, quinone content, and the anticancer activity of NS oil, which could aid in standardization of this important phytomedicinal plant.
Materials and methods
Preparation of plant extract
NS seeds were purchased from a local market, and stored as a voucher specimen. Water and methanol extracts of NS were prepared following exposure to different temperatures (by heating). After the different thermal processing, the seeds were washed, dried, and crushed manually to a powder with a mortar and pestle at room temperature in the absence of sunlight. Next, 15 mL of double-distilled water and 10 mL of hexane (95%), dichloromethane (5%) was added to 5 g of powder, with continuous vortexing until there was no further change in the color of the solution, which was then stored overnight at 4°C in sterile tubes covered by aluminum foil. The extract was centrifuged at 10,000× g for 30 minutes, and the upper oily and lower aqueous phases were separated and transferred to new tubes. The samples were later evaporated under reduced pressure using a rotary evaporator and then filtered through a Minisart filter and refrigerated until use. Precautions were taken to ensure the stability of the analysis samples, which are light-sensitive and heat-sensitive, since quinones of this type readily form radicals when exposed to light. Thus, immediately after preparation, vials containing the seed oil extract were covered by aluminum foil to them protect from light. Under these conditions, the extracts were stable for at least 2 months.12,24
In vitro experiments
The in vitro experiments utilized non-cancerous fibroblasts and two mouse tumor cell lines, ie, Hodgkin’s lymphoma (L428) and colon carcinoma (MC38). The MC38 cells and non-cancerous fibroblasts were maintained in culture using high glucose Dulbecco’s Modified Eagle’s Medium, while the L428 cells were grown in Roswell Park Memorial Institute medium. All media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were in logarithmic growth at the time of use and were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. All drug powder were dissolved in double-distilled water to a concentration of 100 mM or 10 mM and stored at −80°C.
Reverse-phase high-performance liquid chromatography
Reverse-phase high-performance liquid chromatography was used to separate, identify, and quantify the pharmacologically active constituent, TQ, in NS seed extracts heated to different temperatures. Extraction of the constituent from the oil was carried out on a RP-C18 column, using an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v). Samples were filtered through a 0.45 μm filter and deaerated before use. One-minute fractions were collected. Ultraviolet monitoring of the eluted solutes was carried out at 254 nm for TQ. Analyses were performed at ambient temperature at a flow rate of 2 mL per minute.5 Analytic departure time was determined by TQ 99% standard, and the retention time of TQ was 16 minutes under these conditions. Electrospray ionization mass spectrometry of TQ in positive mode was found to be 165.12 [M+H]+.
NF-κB luciferase reporter gene assay
The effects of NS extracts on NF-κB activity were determined using a NF-κB luciferase reporter gene assay. For initial selection of cells, we incubated L428 cells in Roswell Park Memorial Institute medium containing G418 1 mg/mL for 2 weeks. L428 cells (106 per well) expressing the NF-κB luciferase reporter gene were incubated in 1 mL of medium containing 10 μL of methanolic extracts of NS seeds that were preheated to different temperatures for 2 hours. The cells were centrifuged at 2,000 rpm for 5 minutes, after which the supernatant was removed and the pellets were suspended in 70 μL of lysate buffer. The lysates were incubated for 10 minutes at room temperature and centrifuged at 13,000 rpm and 4°C for 1 minute. Next, 20 μL samples from the lysates were mixed with 20 μL of luciferase. The cells were then monitored by a luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Measurements were carried out using a luminometer at 300 nm. The data were normalized to the protein concentration in each lysate as measured by the Bradford method (Bio-Rad, Berkeley, CA, USA).25Nuphar lutea, a water plant that has proven to be a successful inhibitor of NF-κB transcription in cancer cells, was used as a positive control.25 Medium without treatment was used as the corresponding negative control.
In vitro evaluation and role of heating of NS oil on tumor cell proliferation
In vitro experiments were carried out utilizing non-cancerous fibroblasts and a mouse colon carcinoma (MC38) cell line. Figure 1 shows that NS oil did not significantly change the rate of cell growth when compared with the control group, irrespective of thermal processing of the seeds. On the other hand, unheated NS oil decreased the growth rate of MC38 cancer cells by 40%, and heating to 50°C for 10 minutes led to a 90% decrease in the cell growth rate (Figure 1). The IC50 for the unheated NS oil was found to be 1.4 μg/mL, while the heating process yielded higher growth inhibition potency toward MC38 cells, with an IC50 of 0.6 μg/mL.
Figure 1 Effect of Nigella sativa oil extracted from non-heated seeds versus seeds heated to 50°C on growth rate of non-cancerous fibroblasts versus a mouse colon carcinoma (MC38) cell line. Data are presented as the mean ± standard deviation (n=4).
In order to track the behavior of the oil after various thermal processing protocols, NS seeds were heated to different temperatures (no heating, 25°C, 50°C, 100°C, 150°C, 200°C, and 250°C) for 10 minutes, and the effect of the corresponding oil on proliferation of MC38 cancer cells was measured after 24, 48, and 72 hours of incubation (Figure 2). Three different patterns were observed in the different thermal processing protocol, ie, no effect, a mild effect, and a strong effect. Heating the NS seeds to temperatures of 50°C, 100°C, or 150°C produced an oil with a strong ability to inhibit tumor cell growth, while NS oil from seeds without thermal processing (no heating or heating to 25°C) had a mild antiproliferative effect on MC38 cells. NS oil produced from seeds heated to temperatures of 200°C and 250°C did not show any significant effect on tumor cell proliferation. It should be noted that, in all cases, the effect of the different treatments on tumor cell proliferation was steady throughout the 72-hour study period (Figure 2).
Figure 2 Effect of Nigella sativa oil extracted from seeds after different thermal processing protocols (no heating, 25°C, 50°C, 100°C, 150°C, 200°C, and 250°C for 10 minutes) on growth rate of mouse colon carcinoma (MC38) cells following 24, 48, or 72 hours of incubation. Data are presented as the mean ± standard deviation (n=4).
TQ content of NS oil following different thermal processing protocols
Since the biological activity of NS oil is attributed mainly to TQ, we measured the effect of thermal processing of the NS seeds on the TQ content of the corresponding oil (Figure 3). NS seeds heated to 25°C had no effect on the relative amount of TQ when compared with the control group (no heating). The relative TQ content of the oil increased significantly when heated to 50°C, 100°C, or 150°C. In contrast, the extracts derived from seeds after heating to temperatures of 200°C or 250°C showed a significant reduction of TQ content.
Figure 3 Effect of the different thermal processing protocols used for Nigella sativa seeds (no heating, 25°C, 50°C, 100°C, 150°C, 200°C, and 250°C for 10 minutes) on the TQ content of the corresponding oil. Data are presented as the mean ± standard deviation (n=4).
These studies revealed an excellent correlation between the effect of NS oil on tumor cell growth rate and TQ content of the oil for the different thermal processing protocols (Figure 4). In both categories (effect on cell growth rate versus TQ content) a division into three groups was discovered, one with no effect (200°C and 250°C), one with a mild effect (no heating and 25°C), and one with a strong and significant effect (50°C, 100°C, and 150°C). The results show that controlled heating of NS seeds prior to the extraction process causes an increase in the relative TQ content, thereby increases the anticancer activity of the corresponding oil. Back calculation of the yield revealed that one gram of NS oil processed at 50°C yielded 8.3 mg of TQ.
Figure 4 Correlation between effect of Nigella sativa oil on tumor cell growth rate and oil TQ content for the different thermal processing protocols used for the Nigella sativa seeds (no heating, 25°C, 50°C, 100°C, 150°C, 200°C, and 250°C for 10 minutes). Data are presented as the mean ± standard deviation (n=4).
Effect of heated NS seed oil on inhibition of the NF-κB pathway
After determining the relationship between thermal processing of the NS seeds, the TQ content of the oil, and the antiproliferative effect on cancer cells, we investigated the effect of NS oil from heated versus non-heated seeds on the NF-κB signaling pathway (Figure 5). The results show that 2 hours of incubation with NS oil extracted from non-heated seeds resulted in ~50% inhibition, while NS oil from heated seeds caused a complete inhibition in the expression of NF-κB transcription, similar to the positive control (Figure 5).
Figure 5 Effect of Nigella sativa oil from heated (50°C) versus non-heated seeds on the NF-κB signaling pathway in Hodgkin’s lymphoma (L428) cells expressing the NF-κB luciferase reporter gene. Extract of NUP was used as positive control, and medium without additives as negative control. Data are presented as the mean ± standard deviation (n=3).
The main goal of this work was to study the effect of thermal processing of NS seeds on the activity and chemistry profile of the corresponding oil. Previous studies reported that different storage, extraction, and isolation conditions may lead to a marked difference in the amounts of quinone constituents in the oil.5 Following analysis of the results presented here, we posit that controlled heating of the seeds during the preparation process leads to significant structural changes in the active ingredients of the oil, therefore increasing the antiproliferative activity against cancer cells. Most of the biological activity of NS is attributed to the quinone family, including dithymoquinone, THQ, and thymol, in addition to TQ, which are known for their specific anticancer effect in diverse human cancer cell lines.24 This specificity is because activation of NF-κB is significantly higher in cancerous cells than in normal cells, and since the mechanism of action of TQ involves NF-κB (Figure 5), the effect of TQ on cancer cells is much more profound than on normal cells. Studies comparing the anticancer activity and toxicity of various quinone compounds found differences in the degree of their effectiveness against various cancer cell lines. A previous study that examined and compared the anticancer activity of TQ and THQ found that although these compounds are both active in a dose-dependent manner, low doses of THQ were less effective than TQ against cancer cells.24 Following controlled thermal processing of the seeds, the level of TQ in the NS oil increased significantly (Figure 3), which can explain the higher activity of the oil from heated seeds when compared with oil from non-heated seeds.
Considering the chemical composition of the aforementioned quinone compounds, we hypothesize that thermal processing causes chemical changes such as oxidation, leading to modification and conversion between compounds, and accumulation of more potent compounds. We posit that controlled heating causes oxidation of thymol and converts it into THQ. The persistence of the heating process leads to an additional oxidative process that converts THQ into TQ, leading to accumulation of larger amounts of TQ (Figure 6). The photoisomerization of TQ, due to the presence of light, may lead to accumulation of its dimer, dithymoquinone (Figure 6).
Figure 6 Hypothesized mechanism of transition between quinones by an oxidation process after controlled heating of Nigella sativa seeds.
We found that the chemical processes leading to accumulation of TQ occur at temperatures between 50°C and 150°C. Heating the seeds to 200°C and above produces oil with no anticancer effect or ability to inhibit the NF-κB activation pathway. Of note, the boiling point of TQ is 230°C–232°C,25 which explains the significant decrease in anticancer activity and in the relative amount of TQ after heating to high temperatures.
This study, like others in recent years, provides scientific support for the traditional use of NS oil. Our work showed that the cytotoxic activity of NS seed extracts involves dose-dependent inhibition of cell growth and was more active in cancer cells than in non-cancerous cells. It was also found that heating of NS seeds before oil extraction produces oil that is more effective against cancer cells. In addition, our results suggest that heating the seeds before preparation of the extract causes a change in the relative amount of TQ, which explains the differences in their anticancer activity. In conclusion, there is room for further scientific research on NS oil and seeds, including in in vivo models and further clinical trials.
The authors report no conflicts of interest in this work.
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